Chemical imaging has a powerful capability for material characterization, process monitoring, quality control and disease-state determination. Chemical imaging combines chemical analysis with high-resolution optical imaging including optical spectroscopy, Raman imaging as well as fluorescent and IR techniques.
Raman effect is a phenomenon in which a specimen scatters incident light of a given frequency into a spectrum which has lines caused by interaction of the incident light with the molecules making up the specimen. Different molecular species have different characteristic Raman spectra. Consequently, the Raman effect can be used to analyze the molecular species present in the sample. Raman chemical imaging provides molecular-specific image contrast without the use of strains or dies. Raman image contrast arises from a material's intrinsic vibrational spectroscopic signature which is highly sensitive to the composition and structure of the sample as well as its local environment. As a result, Raman imaging can be performed with little or no sample preparation and is widely applicable for material research, failure analysis, process monitoring and clinical diagnosis.
In early Raman analysis, monochromatic light was used as the excitation source and the scattered light from a sample was passed through a monochromator equipped with diffraction grating in order to select a particular line of the resulting Raman spectrum. U.S. Pat. No. 5,194,192 (incorporated herein in its entirety for background information) discloses a Raman analysis apparatus in which the monochromator with diffraction grating is replaced by an imaging camera with a tunable non-dispersive filter, such as a multi-layer dielectric interference filter. Wavelength tuning offered by the filter is fulfilled by rotating it through various small angles of incidence about an axis perpendicular to the optical axis. Because the wavelength selection is a function of the beam's incident angle, there are at least two inherent disadvantages to using a tunable filter. First, interference filters have a small free spectral range. Second, since the imaging beams emanating from a 2D field of view (“FOV”) are not parallel to each other, they do not have the same incident angles and the actual tuned wavelengths at a selected angular position may cover a broad bandpass.
The historical trend in Raman imaging demonstrate that employing means to simultaneously record spatial and Raman spectral information are preferred. As in modern imaging systems, a charge-coupled device (“CCD”) is used to record two dimensions of the three-dimensional information inherent in a Raman image data set. Raman imaging systems can be differentiated by the means they employ to collect the third dimension of information. Conventional Raman imaging systems include dispersive monochromator coupled to CCD's that rely on either 2D point scanning or 1D line scanning as described in U.S. Pat. Nos. 5,048,959 and 6,069,690.
Compared to conventional non-imaging systems, a Raman imaging system based on a tunable filter enables visualizing the distribution (morphology and architecture) of chemical species in heterogeneous samples with molecular compositional specificity. Raman images can be collected non-invasively with limited or no sample preparation at high spatial resolution and with high fidelity where the spatial fidelity is limited by the number of pixels of the CCD detector. Most importantly, every image pixel has associated with it a Raman spectrum whose quality is comparable to that obtained with conventional non-imaging spectrometers. Chemical imaging simultaneously provides image information on the size, shape and distribution (the image morphology) of molecular chemical species present with the sample.
In order to acquire 3D data sets in Raman imaging systems, the two dimensions of the image are recorded directly by a CCD camera while the multispectral information is acquired by capturing images at discrete wavelengths selected by the tunable filter. For this purpose, many techniques suitable for tunable filters have been investigated, among which, two types of tunable filters stand out.
The first type, acousto-optic tunable filters (“AOTFs”), is generally described in U.S. Pat. Nos. 5,377,003, 5,528,368 and RE 36,529. An AOTF Raman imaging system can provide high throughput and broad spectral coverage but it has distinct limitations. For example, such filters suffer from broad spectral bandpass which considerably degrades imaging performance from the diffraction-limited conditions. In effect, AOTFs provide spectral resolution that is an order of magnitude worse than that of a typical Raman spectrometer. Spatial resolution is approximately 2.5 times worse for AOTFs than the diffraction limit.
The second type is the liquid crystal tunable filter (“LCTF”) which is considered a better alternative to the AOTF. In general, LCTF is an electro-optically controllable spectral bandpass filter which can function from the visible region to the near IR with a continuously tunable wavelength. In an imaging system an LCTF is free of image shift with tuning. A nematic LCTF based on the design of the Lyot birefringent filter has been used in a Raman imaging system, as well as Evans Split-Element type LCTF, as disclosed in U.S. Pat. No. 6,002,476. LTCFs have noticeable drawbacks. For example, LCTFs have a low peak transmittance. In addition, LCTFs are susceptible to thermally-induced drift in their spectral bandpass. In theory, the LCTF is free of optical distortions and spectral leakage; but in reality these defects always exist. Finally, LCTF systems are costly.